Methods for obtaining positive transformants of a filamentous fungal host cell

ABSTRACT

The present invention relates to methods for obtaining positive transformants of a filamentous fungal host cell, comprising: transforming a tandem construct into a population of cells of the filamentous fungal host a tandem construct and isolating a transformant of the filamentous fungal host cell comprising the tandem construct. The present invention also relates to such tandem constructs, filamentous fungal host cells comprising such tandem constructs, and methods of producing multiple recombinant proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. §371 national application ofPCT/US2012/052146 filed Aug. 23, 2012, which claims priority or thebenefit under 35 U.S.C. §119 of U.S. Provisional Application No.61/526,804 filed on Aug. 24, 2011, the contents of which are fullyincorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods for increasing the generationof positive transformants of a filamentous fungal host cell expressingmultiple recombinant polypeptides.

2. Description of the Related Art

Recombinant production of a polypeptide in a filamentous fungal hostcell may provide for a more desirable vehicle for producing thepolypeptide in commercially relevant quantities. The recombinantproduction of a polypeptide is generally accomplished by constructing anexpression cassette in which the DNA coding for the polypeptide isplaced under the expression control of a promoter from a regulated gene.The expression cassette is introduced into the host cell, usually byplasmid-mediated transformation. Production of the polypeptide is thenachieved by culturing the transformed host cell under inducingconditions necessary for the proper functioning of the promotercontained on the expression cassette.

Filamentous fungal cells may be transformed with a vector by a processinvolving protoplast formation, transformation of the protoplasts, andregeneration of the cell wall in a manner known per se.Co-transformation of two or more vectors expressing multiple recombinantproteins does not efficiently provide positive transformants producingsignificant amounts of the multiple recombinant polypeptides.

There is a need in the art for methods that improve the efficiency ofobtaining positive transformants producing significant amounts ofmultiple recombinant polypeptides to reduce the number of transformantsto be screened compared to positive transformants generated byco-transformation of vectors for each of the multiple recombinantpolypeptides.

The present invention provides improved methods for the generation ofpositive transformants of a filamentous fungal host cell expressingmultiple recombinant polypeptides.

SUMMARY OF THE INVENTION

The present invention relates to methods for obtaining positivetransformants of a filamentous fungal host cell, comprising:

(a) transforming into a population of cells of the filamentous fungalhost a tandem construct comprising (i) one or more (e.g., several)selectable markers, (ii) a first polynucleotide encoding a firstpolypeptide having biological activity operably linked to a firstpromoter and a first terminator, and (iii) a second polynucleotideencoding a second polypeptide having biological activity operably linkedto a second promoter and a second terminator, wherein the tandemconstruct integrates by ectopic integration;

(b) selecting transformants based on the one or more (e.g., several)selectable markers, wherein the number of positive transformants for thefirst and second polypeptides having biological activity obtained bytransformation of the tandem construct is higher compared to the numberof positive transformants obtained by co-transformation of separateconstructs for each of the first and second polynucleotides; and

(c) isolating a transformant of the filamentous fungal host cellcomprising the tandem construct expressing the first and secondpolypeptides having biological activity.

The present invention also relates to filamentous fungal host cells,comprising: a tandem construct comprising (i) one or more (e.g.,several) selectable markers, (ii) a first polynucleotide encoding afirst polypeptide having biological activity operably linked to a firstpromoter and a first terminator, and (iii) a second polynucleotideencoding a second polypeptide having biological activity operably linkedto a second promoter and a second terminator, wherein the tandemconstruct integrated by ectopic integration.

The present invention also relates to methods of producing multiplerecombinant polypeptides having biological activity, comprising:

(a) cultivating a filamentous fungal host cell transformed with a tandemconstruct comprising (i) one or more (e.g., several) selectable markers,(ii) a first polynucleotide encoding a first polypeptide havingbiological activity operably linked to a first promoter and a firstterminator, and (iii) a second polynucleotide encoding a secondpolypeptide having biological activity operably linked to a secondpromoter and a second terminator, wherein the tandem constructintegrates by ectopic integration, under conditions conducive forproduction of the polypeptides; and optionally

(b) recovering the first and second polypeptides having biologicalactivity.

The present invention further relates to tandem constructs andexpression vectors comprising (i) one or more (e.g., several) selectablemarkers, (ii) a first polynucleotide encoding a first polypeptide havingbiological activity operably linked to a first promoter and a firstterminator, and (iii) a second polynucleotide encoding a secondpolypeptide having biological activity operably linked to a secondpromoter and a second terminator.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a restriction map of plasmid pAG43.

FIG. 2 shows a restriction map of plasmid pSMai214.

FIG. 3 shows a restriction map of plasmid pDM287.

FIGS. 4A-4D shows SDS-PAGE profiles of 45 transformants of pDM287(transformation of tandem construct; 4A and 4B) and 45 transformants ofpEJG107+pSMai214 (co-transformation; 4C and 4D).

FIG. 5 shows a comparison of positive transformants for beta-glucosidaseactivity: between 45 transformants of pDM287 and 45 transformants ofpEJG107+pSMai214.

DEFINITIONS

Acetylxylan esterase: The term “acetylxylan esterase” means acarboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetylgroups from polymeric xylan, acetylated xylose, acetylated glucose,alpha-napthyl acetate, and p-nitrophenyl acetate. For purposes of thepresent invention, acetylxylan esterase activity is determined using 0.5mM p-nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0containing 0.01% TWEEN™ 20 (polyoxyethylene sorbitan monolaurate). Oneunit of acetylxylan esterase is defined as the amount of enzyme capableof releasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 25°C.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Alpha-L-arabinofuranosidase: The term “alpha-L-arabinofuranosidase”means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55)that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeacts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.Alpha-L-arabinofuranosidase is also known as arabinosidase,alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase,polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranosidehydrolase, L-arabinosidase, or alpha-L-arabinanase. For purposes of thepresent invention, alpha-L-arabinofuranosidase activity is determinedusing 5 mg of medium viscosity wheat arabinoxylan (MegazymeInternational Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100mM sodium acetate pH 5 in a total volume of 200 μl for 30 minutes at 40°C. followed by arabinose analysis by AMINEX® HPX-87H columnchromatography (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Alpha-glucuronidase: The term “alpha-glucuronidase” means analpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzesthe hydrolysis of an alpha-D-glucuronoside to D-glucuronate and analcohol. For purposes of the present invention, alpha-glucuronidaseactivity is determined according to de Vries, 1998, J. Bacteriol. 180:243-249. One unit of alpha-glucuronidase equals the amount of enzymecapable of releasing 1 μmole of glucuronic or 4-O-methylglucuronic acidper minute at pH 5, 40° C.

Aspartic protease: The term “aspartic protease” means a protease thatuses an aspartate residue(s) for catalyzing the hydrolysis of peptidebonds in peptides and proteins. Aspartic proteases are a family ofprotease enzymes that use an aspartate residue for catalytic hydrolysisof their peptide substrates. In general, they have two highly-conservedaspartates in the active site and are optimally active at acidic pH(Szecsi, 1992, Scand. J. Clin. Lab. In vest. Suppl. 210: 5-22). Forpurposes of the present invention, aspartic protease activity isdetermined according to the procedure described by Aikawa et al., 2001,J. Biochem. 129: 791-794.

Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminalnon-reducing beta-D-glucose residues with the release of beta-D-glucose.For purposes of the present invention, beta-glucosidase activity isdetermined using p-nitrophenyl-beta-D-glucopyranoside as substrateaccording to the procedure of Venturi et al., 2002, Extracellularbeta-D-glucosidase from Chaetomium thermophilum var. coprophilum:production, purification and some biochemical properties, J. BasicMicrobiol. 42: 55-66. One unit of beta-glucosidase is defined as 1.0μmole of p-nitrophenolate anion produced per minute at 25° C., pH 4.8from 1 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mMsodium citrate containing 0.01% TWEEN® 20.

Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xylosidexylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of shortbeta→(4)-xylooligosaccharides to remove successive D-xylose residuesfrom non-reducing termini. For purposes of the present invention, oneunit of beta-xylosidase is defined as 1.0 μmole of p-nitrophenolateanion produced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citratecontaining 0.01% TWEEN® 20.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1.176)that catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages incellulose, cellooligosaccharides, or any beta-1,4-linked glucosecontaining polymer, releasing cellobiose from the reducing ornon-reducing ends of the chain (Teeri, 1997, Crystalline cellulosedegradation: New insight into the function of cellobiohydrolases, Trendsin Biotechnology 15: 160-167; Teeri et al., 1998, Trichoderma reeseicellobiohydrolases: why so efficient on crystalline cellulose?, Biochem.Soc. Trans. 26: 173-178). Cellobiohydrolase activity is determinedaccording to the procedures described by Lever et al., 1972, Anal.Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBS Letters, 149:152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters, 187: 283-288;and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581. In the presentinvention, the Tomme et al. method can be used to determinecellobiohydrolase activity.

Cellulolytic enzyme or cellulase: The term “cellulolytic enzyme” or“cellulase” means one or more (e.g., several) enzymes that hydrolyze acellulosic material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic activity include: (1)measuring the total cellulolytic activity, and (2) measuring theindividual cellulolytic activities (endoglucanases, cellobiohydrolases,and beta-glucosidases) as reviewed in Zhang et al., Outlook forcellulase improvement: Screening and selection strategies, 2006,Biotechnology Advances 24: 452-481. Total cellulolytic activity isusually measured using insoluble substrates, including Whatman No. 1filter paper, microcrystalline cellulose, bacterial cellulose, algalcellulose, cotton, pretreated lignocellulose, etc. The most common totalcellulolytic activity assay is the filter paper assay using Whatman No.1 filter paper as the substrate. The assay was established by theInternational Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987,Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

For purposes of the present invention, cellulolytic enzyme activity isdetermined by measuring the increase in hydrolysis of a cellulosicmaterial by cellulolytic enzyme(s) under the following conditions: 1-50mg of cellulolytic enzyme protein/g of cellulose in PCS (or otherpretreated cellulosic material) for 3-7 days at a suitable temperature,e.g., 50° C., 55° C., or 60° C., compared to a control hydrolysiswithout addition of cellulolytic enzyme protein. Typical conditions are1 ml reactions, washed or unwashed PCS, 5% insoluble solids, 50 mMsodium acetate pH 5, 1 mM MnSO₄, 50° C., 55° C., or 60° C., 72 hours,sugar analysis by AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc.,Hercules, Calif., USA).

Cellulosic material: The term “cellulosic material” means any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant is hemicellulose,and the third is pectin. The secondary cell wall, produced after thecell has stopped growing, also contains polysaccharides and isstrengthened by polymeric lignin covalently cross-linked tohemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thusa linear beta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which help stabilize the cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, agricultural residue,herbaceous material (including energy crops), municipal solid waste,pulp and paper mill residue, waste paper, and wood (including forestryresidue) (see, for example, Wiselogel et al., 1995, in Handbook onBioethanol (Charles E. Wyman, editor), pp. 105-118, Taylor & Francis,Washington D.C.; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd,1990, Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier etal., 1999, Recent Progress in Bioconversion of Lignocellulosics, inAdvances in Biochemical Engineering/Biotechnology, T. Scheper, managingeditor, Volume 65, pp. 23-40, Springer-Verlag, New York). It isunderstood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix. In a preferred aspect, thecellulosic material is any biomass material. In another preferredaspect, the cellulosic material is lignocellulose, which comprisescellulose, hemicelluloses, and lignin.

In one aspect, the cellulosic material is agricultural residue. Inanother aspect, the cellulosic material is herbaceous material(including energy crops). In another aspect, the cellulosic material ismunicipal solid waste. In another aspect, the cellulosic material ispulp and paper mill residue. In another aspect, the cellulosic materialis waste paper. In another aspect, the cellulosic material is wood(including forestry residue).

In another aspect, the cellulosic material is arundo. In another aspect,the cellulosic material is bagasse. In another aspect, the cellulosicmaterial is bamboo. In another aspect, the cellulosic material is corncob. In another aspect, the cellulosic material is corn fiber. Inanother aspect, the cellulosic material is corn stover. In anotheraspect, the cellulosic material is miscanthus. In another aspect, thecellulosic material is orange peel. In another aspect, the cellulosicmaterial is rice straw. In another aspect, the cellulosic material isswitchgrass. In another aspect, the cellulosic material is wheat straw.

In another aspect, the cellulosic material is aspen. In another aspect,the cellulosic material is eucalyptus. In another aspect, the cellulosicmaterial is fir. In another aspect, the cellulosic material is pine. Inanother aspect, the cellulosic material is poplar. In another aspect,the cellulosic material is spruce. In another aspect, the cellulosicmaterial is willow.

In another aspect, the cellulosic material is algal cellulose. Inanother aspect, the cellulosic material is bacterial cellulose. Inanother aspect, the cellulosic material is cotton linter. In anotheraspect, the cellulosic material is filter paper. In another aspect, thecellulosic material is microcrystalline cellulose. In another aspect,the cellulosic material is phosphoric-acid treated cellulose.

In another aspect, the cellulosic material is an aquatic biomass. Asused herein the term “aquatic biomass” means biomass produced in anaquatic environment by a photosynthesis process. The aquatic biomass canbe algae, emergent plants, floating-leaf plants, or submerged plants.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect, the cellulosic material is pretreated.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding apolypeptide. Each control sequence may be native (i.e., from the samegene) or foreign (i.e., from a different gene) to the polynucleotideencoding the polypeptide or native or foreign to each other. Suchcontrol sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a polypeptide.

Ectopic integration: The term “ectopic integration” means the insertionof a nucleic acid into the genome of a microorganism at a non-targetedsite or at a site other than its usual chromosomal locus, i.e., randomintegration.

Endoglucanase: The term “endoglucanase” means anendo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) thatcatalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose,cellulose derivatives (such as carboxymethyl cellulose and hydroxyethylcellulose), lichenin, beta-1,4 bonds in mixed beta-1,3 glucans such ascereal beta-D-glucans or xyloglucans, and other plant materialcontaining cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) as substrate according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268, at pH 5, 40° C.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Family 61 glycoside hydrolase: The term “Family 61 glycoside hydrolase”or “Family GH61” or “GH61” means a polypeptide falling into theglycoside hydrolase Family 61 according to Henrissat B., 1991, Aclassification of glycosyl hydrolases based on amino-acid sequencesimilarities, Biochem. J. 280: 309-316, and Henrissat B., and BairochA., 1996, Updating the sequence-based classification of glycosylhydrolases, Biochem. J. 316: 695-696. The enzymes in this family wereoriginally classified as a glycoside hydrolase family based onmeasurement of very weak endo-1,4-beta-D-glucanase activity in onefamily member. The structure and mode of action of these enzymes arenon-canonical and they cannot be considered as bona fide glycosidases.However, they are kept in the CAZy classification on the basis of theircapacity to enhance the breakdown of lignocellulose when used inconjunction with a cellulase or a mixture of cellulases.

Feruloyl esterase: The term “feruloyl esterase” means a4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) thatcatalyzes the hydrolysis of 4-hydroxy-3-methoxycinnamoyl (feruloyl)groups from esterified sugar, which is usually arabinose in naturalbiomass substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate).Feruloyl esterase is also known as ferulic acid esterase,hydroxycinnamoyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA,cinnAE, FAE-I, or FAE-II. For purposes of the present invention,feruloyl esterase activity is determined using 0.5 mMp-nitrophenylferulate as substrate in 50 mM sodium acetate pH 5.0. Oneunit of feruloyl esterase equals the amount of enzyme capable ofreleasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 25° C.

Flanking: The term “flanking” means DNA sequences extending on eitherside of a specific DNA sequence, locus, or gene. The flanking DNA isimmediately adjacent to another DNA sequence, locus, or gene that is tobe integrated into the genome of a filamentous fungal cell.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a mature polypeptide main; wherein the fragment has enzymeactivity. In one aspect, a fragment contains at least 85%, e.g., atleast 90% or at least 95% of the amino acid residues of the maturepolypeptide of an enzyme.

Hemicellulolytic enzyme or hemicellulase: The term “hemicellulolyticenzyme” or “hemicellulase” means one or more (e.g., several) enzymesthat hydrolyze a hemicellulosic material. See, for example, Shallom, D.and Shoham, Y. Microbial hemicellulases. Current Opinion InMicrobiology, 2003, 6(3): 219-228). Hemicellulases are key components inthe degradation of plant biomass. Examples of hemicellulases include,but are not limited to, an acetylmannan esterase, an acetylxylanesterase, an arabinanase, an arabinofuranosidase, a coumaric acidesterase, a feruloyl esterase, a galactosidase, a glucuronidase, aglucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and axylosidase. The substrates of these enzymes, the hemicelluloses, are aheterogeneous group of branched and linear polysaccharides that arebound via hydrogen bonds to the cellulose microfibrils in the plant cellwall, crosslinking them into a robust network. Hemicelluloses are alsocovalently attached to lignin, forming together with cellulose a highlycomplex structure. The variable structure and organization ofhemicelluloses require the concerted action of many enzymes for itscomplete degradation. The catalytic modules of hemicellulases are eitherglycoside hydrolases (GHs) that hydrolyze glycosidic bonds, orcarbohydrate esterases (CEs), which hydrolyze ester linkages of acetateor ferulic acid side groups. These catalytic modules, based on homologyof their primary sequence, can be assigned into GH and CE families. Somefamilies, with an overall similar fold, can be further grouped intoclans, marked alphabetically (e.g., GH-A). A most informative andupdated classification of these and other carbohydrate active enzymes isavailable in the Carbohydrate-Active Enzymes (CAZy) database.Hemicellulolytic enzyme activities can be measured according to Ghoseand Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752, at a suitabletemperature, e.g., 50° C., 55° C., or 60° C., and pH, e.g., 5.0 or 5.5.

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

Homologous repeat: The term “homologous repeat” means a fragment of DNAthat is repeated at least twice in the recombinant DNA introduced into ahost cell and which can facilitate the loss of the DNA, i.e., selectablemarker that is inserted between two homologous repeats, by homologousrecombination.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideencoding a polypeptide. The term “host cell” encompasses any progeny ofa parent cell that is not identical to the parent cell due to mutationsthat occur during replication.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., recombinantproduction in a host cell; multiple copies of a gene encoding thesubstance; and use of a stronger promoter than the promoter naturallyassociated with the gene encoding the substance).

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at50° C.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. It is known in the art that a hostcell may produce a mixture of two of more different mature polypeptides(i.e., with a different C-terminal and/or N-terminal amino acid)expressed by the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving enzyme activity.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 60° C.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more (e.g.,several) control sequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Polypeptide having cellulolytic enhancing activity: The term“polypeptide having cellulolytic enhancing activity” means a GH61polypeptide that catalyzes the enhancement of the hydrolysis of acellulosic material by enzyme having cellulolytic activity. For purposesof the present invention, cellulolytic enhancing activity is determinedby measuring the increase in reducing sugars or the increase of thetotal of cellobiose and glucose from the hydrolysis of a cellulosicmaterial by cellulolytic enzyme under the following conditions: 1-50 mgof total protein/g of cellulose in PCS, wherein total protein iscomprised of 50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/wprotein of a GH61 polypeptide having cellulolytic enhancing activity for1-7 days at a suitable temperature, e.g., 50° C., 55° C., or 60° C., andpH, e.g., 5.0 or 5.5, compared to a control hydrolysis with equal totalprotein loading without cellulolytic enhancing activity (1-50 mg ofcellulolytic protein/g of cellulose in PCS). In a preferred aspect, amixture of CELLUCLAST® 1.5 L (Novozymes A/S, Bagsværd, Denmark) in thepresence of 2-3% of total protein weight Aspergillus oryzaebeta-glucosidase (recombinantly produced in Aspergillus oryzae accordingto WO 02/095014) or 2-3% of total protein weight Aspergillus fumigatusbeta-glucosidase (recombinantly produced in Aspergillus oryzae asdescribed in WO 2002/095014) of cellulase protein loading is used as thesource of the cellulolytic activity.

The GH61 polypeptides having cellulolytic enhancing activity enhance thehydrolysis of a cellulosic material catalyzed by enzyme havingcellulolytic activity by reducing the amount of cellulolytic enzymerequired to reach the same degree of hydrolysis preferably at least1.01-fold, e.g., at least 1.05-fold, at least 1.10-fold, at least1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least4-fold, at least 5-fold, at least 10-fold, or at least 20-fold.

Positive transformants: The term “positive transformants” meanstransformants from a population of cells of a filamentous fungal hosttransformed with a tandem construct of the present invention orco-transformed with multiple constructs, wherein the transformantsproduce two or more (e.g., several) recombinant polypeptides encoded bythe tandem construct or the multiple constructs.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the -nobrief option) is usedas the percent identity and is calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the -nobrief option) is used as the percentidentity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having enzyme activity. In one aspect, a subsequence containsat least 85%, e.g., at least 90% or at least 95% of the nucleotides ofthe mature polypeptide coding sequence of an enzyme.

Subtilisin-like serine protease: The term “subtilisin-like serineprotease” means a protease with a substrate specificity similar tosubtilisin that uses a serine residue for catalyzing the hydrolysis ofpeptide bonds in peptides and proteins. Subtilisin-like proteases(subtilases) are serine proteases characterized by a catalytic triad ofthe three amino acids aspartate, histidine, and serine. The arrangementof these catalytic residues is shared with the prototypical subtilisinfrom Bacillus licheniformis (Siezen and Leunissen, 1997, Protein Science6: 501-523). Subtilisin-like serine protease activity can be determinedusing a synthetic substrate,N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (AAPF) (Bachem AG,Bubendorf, Switzerland) in 100 mM NaCl-100 mM MOPS pH 7.0 at 50° C. for3 hours and then the absorbance at 405 nm is measured.

Transformant: The term “transformant” means a cell which has taken upextracellular DNA (foreign, artificial or modified) and expresses thegene(s) contained therein.

Transformation: The term “transformation” means the introduction ofextracellular DNA into a cell, i.e., the genetic alteration of a cellresulting from the direct uptake, incorporation and expression ofexogenous genetic material (exogenous DNA) from its surroundings andtaken up through the cell membrane(s).

Transformation efficiency: The term “transformation efficiency” meansthe efficiency by which cells can take up the extracellular DNA andexpress the gene(s) contained therein, which is calculated by dividingthe number of positive transformants expressing the gene(s) by theamount of DNA used during a transformation procedure.

Trypsin-like serine protease: The term “trypsin-like serine protease”means a protease with a substrate specificity similar to trypsin thatuses a serine residue for catalyzing the hydrolysis of peptide bonds inpeptides and proteins. For purposes of the present invention,trypsin-like serine protease activity is determined according to theprocedure described by Dienes et al., 2007, Enzyme and MicrobialTechnology 40: 1087-1094.

Variant: The term “variant” means a polypeptide having enzyme activitycomprising an alteration, i.e., a substitution, insertion, and/ordeletion, at one or more (e.g., several) positions. A substitution meansreplacement of the amino acid occupying a position with a differentamino acid; a deletion means removal of the amino acid occupying aposition; and an insertion means adding an amino acid adjacent to andimmediately following the amino acid occupying a position.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 70° C.

Very low stringency conditions: The term “very low stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 45° C.

Xylan-containing material: The term “xylan-containing material” meansany material comprising a plant cell wall polysaccharide containing abackbone of beta-(1-4)-linked xylose residues. Xylans of terrestrialplants are heteropolymers possessing a beta-(1-4)-D-xylopyranosebackbone, which is branched by short carbohydrate chains. They compriseD-glucuronic acid or its 4-O-methyl ether, L-arabinose, and/or variousoligosaccharides, composed of D-xylose, L-arabinose, D- or L-galactose,and D-glucose. Xylan-type polysaccharides can be divided into homoxylansand heteroxylans, which include glucuronoxylans,(arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, andcomplex heteroxylans. See, for example, Ebringerova et al., 2005, Adv.Polym. Sci. 186:1-67.

In the processes of the present invention, any material containing xylanmay be used. In a preferred aspect, the xylan-containing material islignocellulose.

Xylan degrading activity or xylanolytic activity: The term “xylandegrading activity” or “xylanolytic activity” means a biologicalactivity that hydrolyzes xylan-containing material. The two basicapproaches for measuring xylanolytic activity include: (1) measuring thetotal xylanolytic activity, and (2) measuring the individual xylanolyticactivities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases). Recent progress in assays of xylanolyticenzymes was summarized in several publications including Biely andPuchard, Recent progress in the assays of xylanolytic enzymes, 2006,Journal of the Science of Food and Agriculture 86(11): 1636-1647;Spanikova and Biely, 2006, Glucuronoyl esterase—Novel carbohydrateesterase produced by Schizophyllum commune, FEBS Letters 580(19):4597-4601; Herrmann, Vrsanska, Jurickova, Hirsch, Biely, and Kubicek,1997, The beta-D-xylosidase of Trichoderma reesei is a multifunctionalbeta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381.

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including, forexample, oat spelt, beechwood, and larchwood xylans, or by photometricdetermination of dyed xylan fragments released from various covalentlydyed xylans. The most common total xylanolytic activity assay is basedon production of reducing sugars from polymeric 4-O-methylglucuronoxylan as described in Bailey, Biely, Poutanen, 1992,Interlaboratory testing of methods for assay of xylanase activity,Journal of Biotechnology 23(3): 257-270. Xylanase activity can also bedetermined with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON®X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) and 200mM sodium phosphate buffer pH 6 at 37° C. One unit of xylanase activityis defined as 1.0 mmole of azurine produced per minute at 37° C., pH 6from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6buffer.

For purposes of the present invention, xylan degrading activity isdetermined by measuring the increase in hydrolysis of birchwood xylan(Sigma Chemical Co., Inc., St. Louis, Mo., USA) by xylan-degradingenzyme(s) under the following typical conditions: 1 ml reactions, 5mg/ml substrate (total solids), 5 mg of xylanolytic protein/g ofsubstrate, 50 mM sodium acetate pH 5, 50° C., 24 hours, sugar analysisusing p-hydroxybenzoic acid hydrazide (PHBAH) assay as described byLever, 1972, A new reaction for colorimetric determination ofcarbohydrates, Anal. Biochem 47: 273-279.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase(E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidiclinkages in xylans. For purposes of the present invention, xylanaseactivity is determined with 0.2% AZCL-arabinoxylan as substrate in 0.01%TRITON® X-100 and 200 mM sodium phosphate buffer pH 6 at 37° C. One unitof xylanase activity is defined as 1.0 μmole of azurine produced perminute at 37° C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200mM sodium phosphate pH 6 buffer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for obtaining positivetransformants of a filamentous fungal host cell, comprising: (a)transforming into a population of cells of the filamentous fungal host atandem construct comprising (i) one or more (e.g., several) selectablemarkers, (ii) a first polynucleotide encoding a first polypeptide havingbiological activity operably linked to a first promoter and a firstterminator, and (iii) a second polynucleotide encoding a secondpolypeptide having biological activity operably linked to a secondpromoter and a second terminator, wherein the tandem constructintegrates by ectopic integration; (b) selecting transformants based onthe one or more (e.g., several) selectable markers, wherein the numberof positive transformants for the first and second polypeptides havingbiological activity obtained by transformation of the tandem constructis higher compared to the number of positive transformants obtained byco-transformation of separate constructs for each of the first andsecond polynucleotides; and (c) isolating a transformant of thefilamentous fungal host cell comprising the tandem construct expressingthe first and second polypeptides having biological activity.

An advantage of the methods of the present invention is an increase inthe transformation efficiency of obtaining positive transformantsproducing significant amounts of two or more (e.g., several) recombinantpolypeptides, which reduces the number of transformants that need to begenerated and screened. Using a tandem construct of the presentinvention expressing two or more recombinant polypeptides results in ahigher number of the transformants producing the two or more recombinantpolypeptides in significant amounts when compared to transformantsgenerated by co-transformation of separate constructs for each of thetwo or more recombinant polypeptides, e.g., two or more individualexpression constructs.

In one aspect, the number of positive transformants for the first andsecond polypeptides having biological activity obtained bytransformation of a tandem construct of the present invention isincreased at least 1.1-fold, e.g., at least 1.25-fold, at least1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least4-fold, at least 5-fold, or at least 10-fold compared to the number ofpositive transformants obtained by co-transformation of separateconstructs for each of the first and second polypeptides havingbiological activity.

Tandem Constructs

The present invention also relates to tandem constructs comprising (i)one or more (e.g., several) selectable markers, (ii) a firstpolynucleotide encoding a first polypeptide having biological activityoperably linked to a first promoter and a first terminator, and (iii) asecond polynucleotide encoding a second polypeptide having biologicalactivity operably linked to a second promoter and a second terminator.The tandem constructs can be constructed by operably linking one or more(e.g., several) control sequences to each polynucleotide of theconstruct that direct the expression of the coding sequence in afilamentous fungal host cell under conditions compatible with thecontrol sequences. Manipulation of each polynucleotide prior toinsertion into a vector may be desirable or necessary depending on theexpression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide that isrecognized by a filamentous fungal host cell for expression of apolynucleotide encoding a polypeptide. The promoter containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the filamentous fungal host cell includingmutant, truncated, and hybrid promoters, and may be obtained from genesencoding extracellular or intracellular polypeptides either homologousor heterologous to the host cell.

In one aspect, the promoters in the tandem constructs are differentpromoters. In another aspect, two or more of the promoters in the tandemconstructs are the same promoter.

Examples of suitable promoters for directing transcription of theconstructs in a filamentous fungal host cell are promoters obtained fromthe genes for Aspergillus nidulans acetamidase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA),Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease,Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporumtrypsin-like protease (WO 96/00787), Fusarium venenatum amyloglucosidase(WO 00/56900), Fusarium venenatum Daria (WO 00/56900), Fusariumvenenatum Quinn (WO 00/56900), Rhizomucor miehei lipase, Rhizomucormiehei aspartic proteinase, Trichoderma reesei beta-glucosidase,Trichoderma reesei cellobiohydrolase I, Trichoderma reeseicellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichodermareesei endoglucanase II, Trichoderma reesei endoglucanase III,Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor, as well as the NA2-tpi promoter (a modified promoterfrom an Aspergillus neutral alpha-amylase gene in which the untranslatedleader has been replaced by an untranslated leader from an Aspergillustriose phosphate isomerase gene; non-limiting examples include modifiedpromoters from an Aspergillus niger neutral alpha-amylase gene in whichthe untranslated leader has been replaced by an untranslated leader froman Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerasegene); and mutant, truncated, and hybrid promoters thereof. Otherpromoters are described in U.S. Pat. No. 6,011,147, which isincorporated herein in its entirety.

The control sequence may also be a transcription terminator, which isrecognized by a filamentous fungal host cell to terminate transcription.The terminator is operably linked to the 3′-terminus of thepolynucleotide encoding the polypeptide. Any terminator that isfunctional in the host cell may be used in the present invention.

In one aspect, the terminators in the tandem constructs are differentterminators. In another aspect, two or more of the terminators in thetandem constructs are the same terminator.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, Fusarium oxysporum trypsin-likeprotease, Trichoderma reesei beta-glucosidase, Trichoderma reeseicellobiohydrolase I, Trichoderma reesei cellobiohydrolase II,Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II,Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanaseV, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II,Trichoderma reesei xylanase III, Trichoderma reesei beta-xylosidase, andTrichoderma reesei translation elongation factor.

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in a filamentous fungal hostcell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by a filamentous fungal host cell as a signalto add polyadenosine residues to transcribed mRNA. Any polyadenylationsequence that is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase Aspergillus oryzae TAKA amylase, Fusarium oxysporumtrypsin-like protease, Trichoderma reesei cellobiohydrolase I,Trichoderma reesei cellobiohydrolase II, and Trichoderma reeseiendoglucanase V.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into a cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a filamentous fungal host cell may be used.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, Rhizomucor mieheiaspartic proteinase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, and Trichoderma reesei endoglucanase V.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Myceliophthorathermophila laccase (WO 95/33836) and Rhizomucor miehei asparticproteinase.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of apolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of a filamentousfungal host cell. Examples of regulatory sequences are those that causeexpression of the gene to be turned on or off in response to a chemicalor physical stimulus, including the presence of a regulatory compound.Regulatory sequences in filamentous fungi include the Aspergillus nigerglucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter,Aspergillus oryzae glucoamylase promoter, Trichoderma reeseicellobiohydrolase I promoter, and Trichoderma reesei cellobiohydrolaseII promoter. Other examples of regulatory sequences are those that allowfor gene amplification. In these cases, the polynucleotide encoding thepolypeptide would be operably linked with the regulatory sequence.

The tandem constructs of the present invention preferably contain one ormore (e.g., several) selectable markers that permit easy selection oftransformed cells. A selectable marker is a gene the product of whichprovides for biocide or viral resistance, resistance to heavy metals,prototrophy to auxotrophs, and the like. Examples of selectable markersfor use in a filamentous fungal host cell include, but are not limitedto, adeA (phosphoribosylaminoimidazole-succinocarboxamide synthase),adeB (phosphoribosylaminoimidazole synthase), amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene. Preferred for use in a Trichodermacell are adeA, adeB, amdS, hph, and pyrG genes. Examples of bacterialselectable markers are markers that confer antibiotic resistance such asampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, ortetracycline resistance.

The one or more (e.g., several) selectable markers may be a dualselectable marker system as described in WO 2010/039889 A2, which isincorporated herein by reference in its entirety. In one aspect, the oneor more (e.g., several) selectable markers is a hph-tk dual selectablemarker system.

In each tandem construct of the present invention, the one or moreselectable markers are different markers, unless a selectable marker isreused as described herein.

One or more (e.g., several) of the selectable markers may be reused forintroducing a new tandem construct into the filamentous fungal hostcell. A tandem construct of the present invention may further comprise afirst homologous repeat flanking 5′ of the one or more (e.g., several)selectable markers and a second homologous repeat flanking 3′ of the oneor more selectable markers, wherein the first homologous repeat and thesecond homologous repeat undergo homologous recombination to excise theone or more selectable markers. Upon the excision of the one or moreselectable markers, the one or more selectable markers can be reused ina new tandem construct.

In one aspect, the first and second homologous repeats are identical. Inanother aspect, the first and second homologous repeats have a sequenceidentity of at least 70%, e.g., at least 75%, at least 80%, at least81%, at least 82%, at least 83% y, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 97%, at least 98%, or at least 99% to eachother. In another aspect, the first and second homologous repeats areeach at least 50 bp, e.g., at least 100 bp, at least 200 bp, at least400 bp, at least 800 bp, at least 1000 bp, at least 1500 bp, or at least2000 bp. The fragment containing one repeat may be longer than thefragment containing the other repeat.

The tandem constructs of the present invention may further comprise oneor more (e.g., several) additional polynucleotides encoding otherpolypeptides having biological activity. For example, a tandem constructmay contain one additional polynucleotide, two additionalpolynucleotides, three additional polynucleotides, etc.

Polypeptides Having Biological Activity

The polypeptides may be any polypeptides having a biological activity ofinterest. The term “polypeptide” is not meant herein to refer to aspecific length of the encoded product and, therefore, encompassespeptides, oligopeptides, and proteins. The term “polypeptide” alsoencompasses two or more polypeptides combined to form the encodedproduct. The polypeptides also include fusion polypeptides, whichcomprise a combination of partial or complete polypeptide sequencesobtained from at least two different polypeptides wherein one or more(e.g., several) may be heterologous to the filamentous fungal host cell.The polypeptides further include naturally occurring allelic andengineered variations of the below-mentioned polypeptides and hybridpolypeptides.

In one aspect, the polypeptides having biological activity may bedifferent polypeptides. In another aspect, two or more of thepolypeptides having biological activity are the same polypeptide.

In another aspect, the polypeptides are selected from the groupconsisting of an antibody, an antigen, an antimicrobial peptide, anenzyme, a growth factor, a hormone, an immunodilator, aneurotransmitter, a receptor, a reporter protein, a structural protein,or a transcription factor.

In another aspect, the enzyme is selected from the group consisting ofan oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase,and a ligase. In another aspect, the enzyme is selected from the groupconsisting of an acetylmannan esterase, acetyxylan esterase,aminopeptidase, alpha-amylase, alpha-galactosidase, alpha-glucosidase,alpha-1,6-transglucosidase, arabinanase, arabinofuranosidase,beta-galactosidase, beta-glucosidase, beta-xylosidase, carbohydrase,carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase,coumaric acid esterase, cyclodextrin glycosyltransferase, cutinase,deoxyribonuclease, endoglucanase, esterase, feruloyl esterase, GH61polypeptide having cellulolytic enhancing activity, glucocerebrosidase,glucose oxidase, glucuronidase, glucuronoyl esterase, haloperoxidase,hemicellulase, invertase, isomerase, laccase, ligase, lipase, mannanase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phospholipase, phytase, phenoloxidase, polyphenoloxidase, proteolyticenzyme, ribonuclease, transglutaminase, urokinase, and xylanase.

In another aspect, the polypeptides are selected from the groupconsisting of an albumin, a collagen, a tropoelastin, an elastin, and agelatin.

In another aspect, the polypeptides are selected from the groupconsisting of a cellulase, a cip1 protein, a GH61 polypeptide havingcellulolytic enhancing activity, a hemicellulase, an esterase, anexpansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin. In another aspect, the cellulase is one ormore enzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase. In another aspect, thehemicellulase is one or more enzymes selected from the group consistingof a xylanase, an acetylxylan esterase, a feruloyl esterase, anarabinofuranosidase, a xylosidase, and a glucuronidase.

In another aspect, one of the polypeptides is a cellulase. In anotheraspect, one of the polypeptides is an endoglucanase. In another aspect,one of the polypeptides is a cellobiohydrolase. In another aspect, oneof the polypeptides is a beta-glucosidase. In another aspect, one of thepolypeptides is a GH61 polypeptide having cellulolytic enhancingactivity. In another aspect, one of the polypeptides is a cip1 protein.In another aspect, one of the polypeptides is an esterase. In anotheraspect, one of the polypeptides is an expansin. In another aspect, oneof the polypeptides is a laccase. In another aspect, one of thepolypeptides is a ligninolytic enzyme. In another aspect, one of thepolypeptides is a pectinase. In another aspect, one of the polypeptidesis a peroxidase. In another aspect, one of the polypeptides is aprotease. In another aspect, one of the polypeptides is a swollenin.

In another aspect, one of the polypeptides is a hemicellulase. Inanother aspect, one of the polypeptides is a xylanase. In anotheraspect, one of the polypeptides is a beta-xylosidase. In another aspect,one of the polypeptides is an acetyxylan esterase. In another aspect,one of the polypeptides is a feruloyl esterase. In another aspect, oneof the polypeptides is an arabinofuranosidase. In another aspect, one ofthe polypeptides is a glucuronidase. In another aspect, one of thepolypeptides is an acetylmannan esterase. In another aspect, one of thepolypeptides is an arabinanase. In another aspect, one of thepolypeptides is a coumaric acid esterase. In another aspect, one of thepolypeptides is a galactosidase. In another aspect, one of thepolypeptides is a glucuronoyl esterase. In another aspect, one of thepolypeptides is a mannanase. In another aspect, one of the polypeptidesis a mannosidase.

Examples of endoglucanases as one of the polypeptides having biologicalactivity, include, but are not limited to, a Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263; Trichodermareesei Cel7B endoglucanase I; GENBANK™ accession no. M15665; SEQ ID NO:2); Trichoderma reesei endoglucanase II (Saloheimo, et al., 1988, Gene63:11-22; Trichoderma reesei Cel5A endoglucanase II; GENBANK™ accessionno. M19373; SEQ ID NO: 4); Trichoderma reesei endoglucanase III (Okadaet al., 1988, Appl. Environ. Microbiol. 64: 555-563; GENBANK™ accessionno. AB003694; SEQ ID NO: 6); Trichoderma reesei endoglucanase V(Saloheimo et al., 1994, Molecular Microbiology 13: 219-228; GENBANK™accession no. Z33381; SEQ ID NO: 8); Aspergillus aculeatus endoglucanase(Ooi et al., 1990, Nucleic Acids Research 18: 5884); Aspergilluskawachii endoglucanase (Sakamoto et al., 1995, Current Genetics 27:435-439); Erwinia carotovara endoglucanase (Saarilahti et al., 1990,Gene 90: 9-14); Fusarium oxysporum endoglucanase (GENBANK™ accession no.L29381); Humicola grisea var. thermoidea endoglucanase (GENBANK™accession no. AB003107); Melanocarpus albomyces endoglucanase (GENBANK™accession no. MAL515703); Neurospora crassa endoglucanase (GENBANK™accession no. XM_324477); Humicola insolens endoglucanase V (SEQ ID NO:10); Myceliophthora thermophila CBS 117.65 endoglucanase (SEQ ID NO:12); basidiomycete CBS 495.95 endoglucanase (SEQ ID NO: 14);basidiomycete CBS 494.95 endoglucanase (SEQ ID NO: 16); Thielaviaterrestris NRRL 8126 CEL6B endoglucanase (SEQ ID NO: 18); Thielaviaterrestris NRRL 8126 CEL6C endoglucanase (SEQ ID NO: 20); Thielaviaterrestris NRRL 8126 CEL7C endoglucanase (SEQ ID NO: 22); Thielaviaterrestris NRRL 8126 CEL7E endoglucanase (SEQ ID NO: 24); Thielaviaterrestris NRRL 8126 CEL7F endoglucanase (SEQ ID NO: 26); Cladorrhinumfoecundissimum ATCC 62373 CEL7A endoglucanase (SEQ ID NO: 28); andTrichoderma reesei strain No. VTT-D-80133 endoglucanase (SEQ ID NO: 30;GENBANK™ accession no. M15665). The endoglucanases of SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, and SEQ ID NO: 30 describedabove are encoded by the mature polypeptide coding sequence of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ IDNO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, and SEQ ID NO:29, respectively.

Examples of cellobiohydrolases as one of the polypeptides havingbiological activity include, but are not limited to, Trichoderma reeseicellobiohydrolase I (SEQ ID NO: 32), Trichoderma reeseicellobiohydrolase II (SEQ ID NO: 34), Humicola insolenscellobiohydrolase I (SEQ ID NO: 36), Myceliophthora thermophilacellobiohydrolase II (WO 2009/042871; SEQ ID NO: 38 and SEQ ID NO: 40),Thielavia terrestris cellobiohydrolase II (CEL6A, WO 2006/074435; SEQ IDNO: 42), Chaetomium thermophilum cellobiohydrolase I (SEQ ID NO: 44),Chaetomium thermophilum cellobiohydrolase II (SEQ ID NO: 46),Aspergillus fumigatus cellobiohydrolase I (SEQ ID NO: 48), andAspergillus fumigatus cellobiohydrolase II (SEQ ID NO: 50). Thecellobiohydrolases of SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46,SEQ ID NO: 48, and SEQ ID NO: 50, described above are encoded by themature polypeptide coding sequence of SEQ ID NO: 31, SEQ ID NO: 33, SEQID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43,SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 49, respectively.

Examples of beta-glucosidases as one of the polypeptides havingbiological activity include, but are not limited to, beta-glucosidasesfrom Aspergillus oryzae (WO 2002/095014; SEQ ID NO: 52), Aspergillusfumigatus (WO 2005/047499; SEQ ID NO: 54), Penicillium brasilianum IBT20888 (WO 2007/019442 and WO 2010/088387; SEQ ID NO: 56), Aspergillusniger (Dan et al., 2000, J. Biol. Chem. 275: 4973-4980; SEQ ID NO: 58),and Aspergillus aculeatus (Kawaguchi et al., 1996, Gene 173: 287-288;SEQ ID NO: 60). The beta-glucosidases of SEQ ID NO: 52, SEQ ID NO: 54,SEQ ID NO: 56, SEQ ID NO: 58, and SEQ ID NO: 60 described above areencoded by the mature polypeptide coding sequence of SEQ ID NO: 51, SEQID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, and SEQ ID NO: 59,respectively.

The beta-glucosidase may also be a fusion protein. In one aspect, thebeta-glucosidase is an Aspergillus oryzae beta-glucosidase variant BGfusion protein (WO 2008/057637; SEQ ID NO: 62) or an Aspergillus oryzaebeta-glucosidase fusion protein (WO 2008/057637; SEQ ID NO: 64). Thebeta-glucosidase fusion proteins of SEQ ID NO: 62 and SEQ ID NO: 64 areencoded by SEQ ID NO: 61 and SEQ ID NO: 63, respectively.

Examples of other endoglucanases, cellobiohydrolases, andbeta-glucosidases are disclosed in numerous Glycosyl Hydrolase familiesusing the classification according to Henrissat B., 1991, Aclassification of glycosyl hydrolases based on amino-acid sequencesimilarities, Biochem. J. 280: 309-316, and Henrissat B., and BairochA., 1996, Updating the sequence-based classification of glycosylhydrolases, Biochem. J. 316: 695-696.

Other cellulolytic enzymes that may be used in the present invention aredescribed in WO 98/13465, WO 98/015619, WO 98/015633, WO 99/06574, WO99/10481, WO 99/025847, WO 99/031255, WO 2002/101078, WO 2003/027306, WO2003/052054, WO 2003/052055, WO 2003/052056, WO 2003/052057, WO2003/052118, WO 2004/016760, WO 2004/043980, WO 2004/048592, WO2005/001065, WO 2005/028636, WO 2005/093050, WO 2005/093073, WO2006/074005, WO 2006/117432, WO 2007/071818, WO 2007/071820, WO2008/008070, WO 2008/008793, U.S. Pat. No. 5,457,046, U.S. Pat. No.5,648,263, and U.S. Pat. No. 5,686,593.

Examples of GH61 polypeptides having cellulolytic enhancing activity asone of the polypeptides having biological activity include, but are notlimited to, GH61 polypeptides from Thielavia terrestris (WO 2005/074647,WO 2008/148131, and WO 2011/035027), Thermoascus aurantiacus (WO2005/074656 and WO 2010/065830), Trichoderma reesei (WO 2007/089290),Myceliophthora thermophila (WO 2009/085935, WO 2009/085859, WO2009/085864, WO 2009/085868), Aspergillus fumigatus (WO 2010/138754),GH61 polypeptides from Penicillium pinophilum (WO 2011/005867),Thermoascus sp. (WO 2011/039319), Penicillium sp. (WO 2011/041397), andThermoascus crustaceous (WO 2011/041504). In one aspect, the GH61polypeptides having cellulolytic enhancing activity include, but are notlimited to, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72,SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO:82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ IDNO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO:110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:128, SEQ ID NO: 130, SEQ ID NO: 132; SEQ ID NO: 134, SEQ ID NO: 136, SEQID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO:146, SEQ ID NO: 148, SEQ ID NO: 150, or SEQ ID NO: 152, or the maturepolypeptide thereof. The GH61 polypeptides described above are encodedby the mature polypeptide coding sequence of SEQ ID NO: 65, SEQ ID NO:67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ IDNO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95,SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO:105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO:123, SEQ ID NO: 125, or SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131,SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ IDNO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149,or SEQ ID NO: 151, respectively.

Examples of xylanases as one of the polypeptides having biologicalactivity include, but are not limited to, xylanases from Aspergillusaculeatus (GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus (WO2006/078256; SEQ ID NO: 154, SEQ ID NO: 156, and SEQ ID NO: 158,Penicillium pinophilum (WO 2011/041405), Penicillium sp. (WO2010/126772), Thielavia terrestris NRRL 8126 (WO 2009/079210), andTrichophaea saccata GH10 (WO 2011/057083). The xylanases described aboveare encoded by the mature polypeptide coding sequence of SEQ ID NO: 153,SEQ ID NO: 155, and SEQ ID NO: 157, respectively.

Examples of beta-xylosidases as one of the polypeptides havingbiological activity include, but are not limited to, beta-xylosidasesfrom Neurospora crassa (SwissProt accession number Q7SOW4), Trichodermareesei (UniProtKB/TrEMBL accession number Q92458; SEQ ID NO: 160),Aspergillus fumigatus (SEQ ID NO; 162), and Talaromyces emersonii(SwissProt accession number Q8×212). The beta-xylosidases describedabove are encoded by the mature polypeptide coding sequence of SEQ IDNO: 159 and SEQ ID NO: 161, respectively.

Examples of acetylxylan esterases as one of the polypeptides havingbiological activity include, but are not limited to, acetylxylanesterases from Aspergillus aculeatus (WO 2010/108918), Chaetomiumglobosum (Uniprot accession number Q2GWX4), Chaetomium gracile (GeneSeqPaccession number AAB82124), Humicola insolens DSM 1800 (WO 2009/073709),Hypocrea jecorina (WO 2005/001036), Myceliophtera thermophila (WO2010/014880), Neurospora crassa (UniProt accession number q7s259),Phaeosphaeria nodorum (Uniprot accession number Q0UHJ1), and Thielaviaterrestris NRRL 8126 (WO 2009/042846).

Examples of feruloyl esterases (ferulic acid esterases) as one of thepolypeptides having biological activity include, but are not limited to,feruloyl esterases form Humicola insolens DSM 1800 (WO 2009/076122),Neosartorya fischeri (UniProt Accession number A1D9T4), Neurosporacrassa (UniProt accession number Q9HGR3), Penicillium aurantiogriseum(WO 2009/127729), and Thielavia terrestris (WO 2010/053838 and WO2010/065448).

Examples of arabinofuranosidases as one of the polypeptides havingbiological activity include, but are not limited to,arabinofuranosidases from Aspergillus niger (GeneSeqP accession numberAAR94170), Humicola insolens DSM 1800 (WO 2006/114094 and WO2009/073383), and M. giganteus (WO 2006/114094).

Examples of alpha-glucuronidases as one of the polypeptides havingbiological activity include, but are not limited to,alpha-glucuronidases from Aspergillus clavatus (UniProt accession numberalcc12), Aspergillus fumigatus (SwissProt accession number Q4WW45),Aspergillus niger (Uniprot accession number Q96WX9), Aspergillus terreus(SwissProt accession number Q0CJP9), Humicola insolens (WO 2010/014706),Penicillium aurantiogriseum (WO 2009/068565), Talaromyces emersonii(UniProt accession number Q8×211), and Trichoderma reesei (Uniprotaccession number Q99024).

The accession numbers are incorporated herein by reference in theirentirety.

Expression Vectors

The present invention also relates to expression vectors comprising atandem construct of the present invention. A tandem construct may beinserted into a vector or the various components of a tandem constructmay be joined together to produce a recombinant expression vector. Thevector may include one or more (e.g., several) convenient restrictionsites to allow for insertion of polynucleotides at such sites. Increating the expression vector, the coding sequences are located in thevector so that the coding sequences are operably linked with theappropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotides. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector preferably contains one or more (e.g., several) selectablemarkers that permit easy selection of transformed cells. Examples ofselectable markers for use in a filamentous fungal host cell include,but are not limited to, adeA(phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB(phosphoribosylaminoimidazole synthase), amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene. Preferred for use in a Trichodermacell are adeA, adeB, amdS, hph, and pyrG genes. Examples of bacterialselectable markers are markers that confer antibiotic resistance such asampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, ortetracycline resistance.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors are well known to one skilled in theart (see, e.g., Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, 2d edition, Cold Spring Harbor, N.Y.).

Filamentous Fungal Host Cells

The present invention also relates to filamentous fungal host cells,comprising: a tandem construct comprising (i) one or more (e.g.,several) selectable markers, (ii) a first polynucleotide encoding afirst polypeptide having biological activity operably linked to a firstpromoter and a first terminator, and (iii) a second polynucleotideencoding a second polypeptide having biological activity operably linkedto a second promoter and a second terminator, wherein the tandemconstruct integrated by ectopic integration.

The tandem construct or an expression vector comprising the tandemconstruct is introduced into a filamentous fungal host cell so that theconstruct is maintained as a chromosomal integrant. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any filamentous fungal cell useful in therecombinant production of polypeptides. “Filamentous fungi” include allfilamentous forms of the subdivision Eumycota and Oomycota (as definedby Hawksworth et al., 1995, supra). The filamentous fungi are generallycharacterized by a mycelial wall composed of chitin, cellulose, glucan,chitosan, mannan, and other complex polysaccharides. Vegetative growthis by hyphal elongation and carbon catabolism is obligately aerobic. Incontrast, vegetative growth by yeasts such as Saccharomyces cerevisiaeis by budding of a unicellular thallus and carbon catabolism may befermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

In one aspect, the filamentous fungal host cell is Aspergillus oryzae.In another aspect, the filamentous fungal host cell is Aspergillusniger. In another aspect, the filamentous fungal host cell is Fusariumvenenatum. In another aspect, the filamentous fungal host cell isTrichoderma reesei. In another aspect, the filamentous fungal host cellis Trichoderma longibrachiatum.

In another aspect, the filamentous fungal host cell is Trichodermareesei RutC30. In another aspect, the filamentous fungal host cell isTrichoderma reesei TV10. In another aspect, the filamentous fungal hostcell is a mutant of Trichoderma reesei RutC30. In another aspect, thefilamentous fungal host cell is a mutant of Trichoderma reesei TV10. Inanother aspect, the filamentous fungal host cell is a morphologicalmutant of Trichoderma reesei. See, for example, WO 97/26330, which isincorporated herein by reference in its entirety.

In another aspect, the filamentous fungal host cell is a Trichodermastrain comprising one or more (e.g., several) genes selected from thegroup consisting of a first subtilisin-like serine protease gene, afirst aspartic protease gene, a trypsin-like serine protease gene, asecond subtilisin-like serine protease gene, and a second asparticprotease gene, wherein the one or more (e.g., several) genes aremodified rendering the mutant strain deficient in the production of oneor more (e.g., several) enzymes selected from the group consisting of afirst subtilisin-like serine protease, a first aspartic protease, atrypsin-like serine protease, a second subtilisin-like serine protease,and a second aspartic protease, respectively, compared to the parentTrichoderma strain when cultivated under identical conditions, asdescribed in WO 2011/075677, which is incorporated herein by referencein its entirety.

Filamentous fungal cells may be transformed by a process involvingprotoplast formation, transformation of the protoplasts, andregeneration of the cell wall in a manner known per se. Suitableprocedures for transformation of Aspergillus and Trichoderma host cellsare described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci.USA 81: 1470-1474, and Christensen et al., 1988, Bio/Technology 6:1419-1422. Suitable methods for transforming Fusarium species aredescribed by Malardier et al., 1989, Gene 78: 147-156, and WO1996/00787.

Methods of Production

The present invention also relates to methods of producing multiplerecombinant polypeptides having biological activity, comprising:

(a) cultivating a filamentous fungal host cell transformed with a tandemconstruct comprising (i) one or more (e.g., several) selectable markers,(ii) a first polynucleotide encoding a first polypeptide havingbiological activity operably linked to a first promoter and a firstterminator, and (iii) a second polynucleotide encoding a secondpolypeptide having biological activity operably linked to a secondpromoter and a second terminator, wherein the tandem constructintegrates by ectopic integration, under conditions conducive forproduction of the polypeptides; and optionally

(b) recovering the first and second polypeptides having biologicalactivity.

The filamentous fungal host cells are cultivated in a nutrient mediumsuitable for production of the polypeptides using methods known in theart. For example, the cells may be cultivated by shake flaskcultivation, or small-scale or large-scale fermentation (includingcontinuous, batch, fed-batch, or solid state fermentations) inlaboratory or industrial fermentors in a suitable medium and underconditions allowing the polypeptides to be expressed and/or isolated.The cultivation takes place in a suitable nutrient medium comprisingcarbon and nitrogen sources and inorganic salts, using procedures knownin the art. Suitable media are available from commercial suppliers ormay be prepared according to published compositions (e.g., in cataloguesof the American Type Culture Collection). If the polypeptides aresecreted into the nutrient medium, the polypeptides can be recovereddirectly from the medium. If the polypeptides are not secreted, they canbe recovered from cell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods include, but arenot limited to, use of specific antibodies, formation of an enzymeproduct, or disappearance of an enzyme substrate. For example, enzymeassays may be used to determine the activity of the polypeptides.

The polypeptides may be recovered using methods known in the art. Forexample, the polypeptides may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation. In one aspect, the whole fermentation broth is recovered.

The polypeptides may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure polypeptides.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Strain

Trichoderma reesei strain 981-O-8 (D4) is a mutagenized strain ofTrichoderma reesei RutC30 (ATCC 56765; Montenecourt and Eveleigh, 1979,Adv. Chem. Ser. 181: 289-301).

Media and Buffer Solutions

2XYT plus ampicillin plates were composed of 16 g of tryptone, 10 g ofyeast extract, 5 g of sodium chloride, 15 g of Bacto agar, and deionizedwater to 1 liter. One ml of a 100 mg/ml solution of ampicillin was addedafter the autoclaved medium was cooled to 55° C.

COVE salt solution was composed of 26 g of KCl, 26 g of MgSO₄.7H₂O, 76 gof KH₂PO₄, 50 ml of COVE trace metals solution, and deionized water to 1liter.

COVE trace metals solution was composed of 0.04 g of NaB₄O₇.10H₂O, 0.4 gof CuSO₄.5H₂O, 1.2 g of FeSO₄.7H₂O, 0.7 g of MnSO₄.H₂O, 0.8 g ofNa₂MoO₂.2H₂O, 10 g of ZnSO₄.7H₂O, and deionized water to 1 liter.

COVE plates were composed of 342.3 g of sucrose, 20 ml of COVE saltsolution, 10 ml of 1 M acetamide, 10 ml of 1.5 M CsCl, 25 g of Nobleagar (Difco), and deionized water to 1 liter.

COVE2 plates were composed of 30 g of sucrose, 20 ml of COVE saltsolution, 10 ml of 1 M acetamide, 25 g of Noble agar (Difco), anddeionized water to 1 liter.

Trichoderma trace metals solution was composed of 216 g of FeCl₃.6H₂O,58 g of ZnSO₄.7H₂O, 27 g of MnSO₄.H₂O, 10 g of CuSO₄.5H₂O, 2.4 g ofH₃BO₃, 336 g of citric acid, and deionized water to 1 liter.

CIM medium was composed of 20 g of cellulose, 10 g of corn steep solids,1.45 g of (NH₄)₂SO₄, 2.08 g of KH₂PO₄, 0.28 g of CaCl₂, 0.42 g ofMgSO₄.7H₂O, 0.42 ml of Trichoderma trace metals solution, 1-2 drops ofantifoam, and deionized water to 1 liter; pH adjusted to 6.0.

YP medium was composed of 10 g of yeast extract, 20 g of Bacto peptone,and deionized water to 1 liter.

PEG buffer was composed of 500 g of polyethylene glycol 4000 (PEG 4000),10 mM CaCl₂, 10 mM Tris-HCl pH 7.5, and deionized water to 1 liter;filter sterilized.

STC was composed of 1 M sorbitol, 10 mM mM CaCl₂, and 10 mM Tris-HCl, pH7.5; filter sterilized.

Example 1 Cloning of an Aspergillus fumigatus GH61B Polypeptide Gene

A tblastn search (Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402) of the Aspergillus fumigatus partial genome sequence (TheInstitute for Genomic Research, Rockville, Md., USA) was performed usingas query several known GH61 polypeptides including the Thermoascusaurantiacus GH61A polypeptide (GeneSeqP Accession Number AEC05922).Several genes were identified as putative Family GH61 homologs basedupon a high degree of similarity to the query sequences at the aminoacid level. One genomic region of approximately 850 bp with greater than70% sequence identity to the Thermoascus aurantiacus GH61A polypeptidesequence at the amino acid level was chosen for further study.

A. fumigatus NN051616 was grown and harvested as described in U.S. Pat.No. 7,244,605. Frozen mycelia were ground, by mortar and pestle, to afine powder and genomic DNA was isolated using a DNEASY® Plant Maxi Kit(QIAGEN Inc., Valencia, Calif., USA) according to manufacturer'sinstructions.

Two synthetic oligonucleotide primers shown below were designed to PCRamplify the A. fumigatus GH61B polypeptide coding sequence from thegenomic DNA. An IN-FUSION® Cloning Kit (Clontech Laboratories, Inc.,Mountain View, Calif., USA) was used to clone the fragment directly intothe expression vector pAlLo2 (WO 2004/099228), without the need forrestriction digestion and ligation.

Forward primer: (SEQ ID NO: 163)5′-ACTGGATTTACCATGACTTTGTCCAAGATCACTTCCA-3′ Reverse primer:(SEQ ID NO: 164) 5′-TCACCTCTAGTTAATTAAGCGTTGAACAGTGCAGGACCAG-3′Bold letters represent coding sequence. The remaining sequences arehomologous to the insertion sites of pAlLo2.

Fifty picomoles of each of the primers above were used in a PCR reactioncomposed of 204 ng of A. fumigatus genomic DNA, 1×Pfx AmplificationBuffer (Invitrogen, Carlsbad, Calif., USA), 1.5 μl of a 10 mM blend ofdATP, dTTP, dGTP, and dCTP, 2.5 units of PLATINUM® Pfx DNA Polymerase(Invitrogen Corp., Carlsbad, Calif., USA), and 1 μl of 50 mM MgSO₄ in afinal volume of 50 μl. The amplification was performed using anEPPENDORF® MASTERCYCLER® 5333 epgradient S (Eppendorf Scientific, Inc.,Westbury, N.Y., USA) programmed for 1 cycle at 94° C. for 3 minutes; and30 cycles each at 94° C. for 30 seconds, 56° C. for 30 seconds, and 72°C. for 1 minutes. The heat block was then held at 72° C. for 15 minutesfollowed by a 4° C. soak cycle. The reaction products were isolated by1.0% agarose gel electrophoresis using 40 mM Tris base, 20 mM sodiumacetate, 1 mM disodium EDTA (TAE) buffer where an approximately 850 bpproduct band was excised from the gel and purified using a MINELUTE® GelExtraction Kit (QIAGEN Inc., Valencia, Calif., USA) according to themanufacturer's instructions.

The 850 bp fragment was then cloned into pAlLo2 using an IN-FUSION®Cloning Kit. Plasmid pAlLo2 was digested with Nco I and Pac I. Theplasmid fragment was purified by gel electrophoresis as above and aQIAQUICK® Gel Purification Kit (QIAGEN Inc., Valencia, Calif., USA). Thegene fragment and the digested vector were combined together in areaction described below resulting in the expression plasmid pAG43(FIG. 1) in which transcription of the A. fumigatus GH61B polypeptidecoding sequence was under the control of the NA2-tpi promoter. TheNA2-tpi promoter is a modified promoter from the Aspergillus nigerneutral alpha-amylase gene in which the untranslated leader has beenreplaced by an untranslated leader from the Aspergillus nidulans triosephosphate isomerase gene. The recombination reaction (20 μl) wascomposed of 1× IN-FUSION® Reaction Buffer (Clontech Laboratories, Inc.,Mountain View, Calif., USA), 1×BSA (Clontech Laboratories, Inc.,Mountain View, Calif., USA), 1 μl of IN-FUSION® Enzyme (diluted 1:10)(Clontech Laboratories, Inc., Mountain View, Calif., USA), 166 ng ofpAlLo2 digested with Nco I and Pac I, and 110 ng of the A. fumigatusGH61B polypeptide purified PCR product. The reaction was incubated at37° C. for 15 minutes followed by 15 minutes at 50° C. The reaction wasdiluted with 40 μl of 10 mM Tris-0.1 M EDTA buffer and 2.5 μl of thediluted reaction was used to transform E. coli XL10 SOLOPACK® GoldCompetent Cells (Stratagene, La Jolla, Calif., USA) according to themanufacturer's instructions. An E. coli transformant containing pAG43(GH61B polypeptide coding sequence) was identified by restriction enzymedigestion and plasmid DNA was prepared using a BIOROBOT® 9600 (QIAGENInc., Valencia, Calif., USA).

DNA sequencing of the 862 bp PCR fragment was performed with an AppliedBiosystems Model 377 XL Automated DNA Sequencer (Applied Biosystems,Carlsbad, Calif., USA) using dye-terminator chemistry (Giesecke et al.,1992, Journal of Virology Methods 38: 47-60) and primer walkingstrategy. The following vector specific primers were used forsequencing:

pAllo2 5 Seq: (SEQ ID NO: 165) 5′-TGTCCCTTGTCGATGCG 3′ pAllo2 3 Seq:(SEQ ID NO: 166) 5′-CACATGACTTGGCTTCC 3′

Nucleotide sequence data were scrutinized for quality and all sequenceswere compared to each other with assistance of PHRED/PHRAP software(University of Washington, Seattle, Wash., USA).

A gene model for the A. fumigatus sequence was constructed based onsimilarity of the encoded protein to the Thermoascus aurantiacus GH61Aprotein (GeneSeqP Accession Number AEC05922). The nucleotide sequenceand deduced amino acid sequence of the A. fumigatus GH61B polypeptidecoding sequence are shown in SEQ ID NO: 93 (DNA sequence) and SEQ ID NO:94 (deduced amino acid sequence). The genomic fragment encodes apolypeptide of 250 amino acids, interrupted by 2 introns of 53 and 56bp. The % G+C content of the coding sequence and the mature codingsequence are 53.9% and 57%, respectively. Using the SignalP softwareprogram (Nielsen et al., 1997, Protein Engineering 10:1-6), a signalpeptide of 21 residues was predicted. The predicted mature proteincontains 221 amino acids with a predicted molecular mass of 23.39 kDa.

Example 2 Construction of pSMai214 for Expression of the Aspergillusfumigatus GH61B Polypeptide

The Aspergillus fumigatus GH61B polypeptide coding sequence wasamplified from plasmid pAG43 (Example 1) using the gene-specific forwardand reverse primers shown below. The region in italics represents vectorhomology to the site of insertion for an IN-FUSION® reaction.

Forward primer: (SEQ ID NO: 167)5′-GGACTGCGCACCATGACTTTGTCCAAGATCACTTCCA-3′ Reverse primer:(SEQ ID NO: 168) 5′-GCCACGGAGCTTAATTAATTAAGCGTTGAACAGTGCAG-3′

Fifty picomoles of each of the primers above were used in a PCR reactioncomposed of 10 ng of pAG43 DNA, 1×Pfx Amplification Buffer, 1.5 μl of a10 mM blend of dATP, dTTP, dGTP, and dCTP, 2.5 units of PLATINUM® PfxDNA Polymerase, and 1 μl of 50 mM MgSO₄ in a final volume of 50 μl. Theamplification was performed using an EPPENDORF®MASTERCYCLER® 5333epgradient S programmed for 1 cycle at 98° C. for 3 minutes; and 30cycles each at 98° C. for 30 seconds, 56° C. for 30 seconds, and 72° C.for 1 minute. The heat block was then held at 72° C. for 15 minutes. ThePCR products were separated by 1% agarose gel electrophoresis using TAEbuffer where an approximately 0.9 kb fragment was excised from the geland extracted using a MINELUTE® Gel Extraction Kit according to themanufacturer's protocol.

Plasmid pMJ09 (WO 2005/047499) was digested with Nco I and Pac I,isolated by 1.0% agarose gel electrophoresis in 1 mM disodium EDTA-50 mMTris base-50 mM boric acid (TBE) buffer, excised from the gel, andextracted using a QIAQUICK® Gel Extraction Kit (QIAGEN Inc., Valencia,Calif., USA) according to the manufacturer's instructions.

The 0.9 kb PCR product was inserted into the gel-purified Nco I/Pac Idigested pMJ09 using an IN-FUSION® PCR Cloning Kit according to themanufacturer's protocol. The IN-FUSION® reaction was composed of 1×IN-FUSION® Reaction Buffer, 100 ng of the gel-purified Nco I/Pac Idigested pMJ09, 37 ng of the 0.9 kb PCR product, 2 μl of 500 μg/ml BSA,and 1 μl of IN-FUSION® Enzyme in a 20 μl reaction volume. The reactionwas incubated for 15 minutes at 37° C. and 15 minutes at 50° C. Afterthe incubation period 30 μl of TE buffer were added to the reaction. A2.5 μl aliquot was used to transform SOLOPACK® Gold Supercompetent Cells(Agilent Technologies, Inc., Cedar Creek, Tex., USA) according to themanufacturer's protocol. Transformants were screened by sequencing andone clone containing the insert with no PCR errors was identified anddesignated pSMai214 (FIG. 2). Plasmid pSMai214 can be digested with PmeI to generate an approximately 5.4 kb fragment for T. reeseitransformation. The 5.4 kb fragment contains the expression cassettecomposed of the T. reesei Cel7A cellobiohydrolase I gene promoter, A.fumigatus GH61B polypeptide coding sequence, T. reesei Cel7Acellobiohydrolase I gene terminator, and Aspergillus nidulansacetamidase (amdS) gene.

Example 3 Construction of a Tandem Construct pDM287 for Expression ofBoth Aspergillus Fumigatus CEL3A Beta-Glucosidase and Aspergillusfumigatus GH61B Polypeptide

An A. fumigatus GH61B polypeptide expression cassette was amplified fromplasmid pSMai214 using the gene-specific forward and reverse primersshown below. The region in italics represents vector homology to thesite of insertion for an IN-FUSION® reaction.

Forward primer: (SEQ ID NO: 169)5′-CGCGGTAGTGGCGCGGTCGACCGAATGTAGGATTGTT-3′ Reverse primer:(SEQ ID NO: 170) 5′-TTACCAATTGGCGCGCCACTACCGCGTTCGAGAAGA-3′

Fifty picomoles of each of the primers above were used in a PCR reactioncomposed of 25 ng of pSMai214 DNA, 1× PHUSION™ High-Fidelity Hot StartDNA Polymerase Buffer (Finnzymes Oy, Espoo, Finland), 1 μl of a 10 mMblend of dATP, dTTP, dGTP, and dCTP, and 1 unit of PHUSION™High-Fidelity Hot Start DNA Polymerase (Finnzymes Oy, Espoo, Finland) ina final volume of 50 μl. The amplification was performed using anEPPENDORF® MASTERCYCLER® 5333 epgradient S programmed for 1 cycle at 98°C. for 30 seconds; 35 cycles each at 98° C. for 10 seconds, 60° C. for30 seconds, and 72° C. for 1 minute 30 seconds; and 1 cycle at 72° C.for 10 minutes. PCR products were separated by 0.8% agarose gelelectrophoresis using TAE buffer where an approximately 2.3 kb fragmentwas excised from the gel and extracted using a NUCLEOSPIN® Extract IIKit (Macherey-Nagel, Inc., Bethlehem, Pa., USA) according to themanufacturer's protocol.

The approximately 2.3 kb PCR product was inserted into Asc I-digestedpEJG107 (WO 2005/047499) using an IN-FUSION® Advantage PCR Cloning Kit(Clontech Laboratories, Inc., Mountain View, Calif., USA) according tothe manufacturer's protocol. Plasmid pEJG107 comprises an Aspergillusfumigatus CEL3A beta-glucosidase encoding sequence (SEQ ID NO: 53 [DNAsequence] and SEQ ID NO: 54 [deduced amino acid sequence]). TheIN-FUSION® reaction was composed of 1×IN-FUSION® Reaction Buffer, 125 ngof the Asc I-digested pEJG107, 90 ng of the 2.33 kb PCR product, and 1μl of IN-FUSION® Enzyme in a 10 μl reaction volume. The reaction wasincubated for 15 minutes at 37° C. followed by 15 minutes at 50° C.After the incubation period 40 μl of TE were added to the reaction. A 2μl aliquot was used to transform ONE SHOT® TOP10 competent cells(Invitrogen, Carlsbad, Calif., USA) according to the manufacturer'sprotocol. The E. coli transformation reactions were spread onto 2XYTplus ampicillin plates. The transformants were screened by sequencingand one clone containing the insert with no PCR errors was identifiedand designated pDM287 (FIG. 3). Plasmid pDM287 can be digested with PmeI to generate an approximately 9.9 kb fragment for T. reeseitransformation. The 9.9 kb fragment contains two expression cassettescomposed of (1) the T. reesei Cel7A cellobiohydrolase I gene promoter,A. fumigatus CEL3A beta-glucosidase coding sequence, and T. reesei Cel7Acellobiohydrolase I gene terminator; and (2) the T. reesei Cel7Acellobiohydrolase I gene promoter, A. fumigatus GH61B polypeptide codingsequence, and T. reesei Cel7A cellobiohydrolase I gene terminator. The9.9 kb fragment also contains the Aspergillus nidulans acetamidase(amdS) gene.

Example 4 Trichoderma Reesei Protoplast Generation and Transformation

Protoplast preparation and transformation were performed using amodified protocol by Penttila et al., 1987, Gene 61: 155-164. Briefly,Trichoderma reesei strain 981-O-8 (D4) was cultivated in 25 ml of YPmedium supplemented with 2% (w/v) glucose and 10 mM uridine at 27° C.for 17 hours with gentle agitation at 90 rpm. Mycelia were collected byfiltration using a Vacuum Driven Disposable Filtration System(Millipore, Bedford, Mass., USA) and washed twice with deionized waterand twice with 1.2 M sorbitol. Protoplasts were generated by suspendingthe washed mycelia in 20 ml of 1.2 M sorbitol containing 15 mg ofGLUCANEX® 200 G (Novozymes A/S, Bagsvaerd, Denmark) per ml and 0.36units of chitinase (Sigma Chemical Co., St. Louis, Mo., USA) per ml for15-25 minutes at 34° C. with gentle shaking at 90 rpm. Protoplasts werecollected by centrifuging for 7 minutes at 400×g and washed twice withcold 1.2 M sorbitol. The protoplasts were counted using a haemocytometerand resuspended to a final concentration of 1×10⁸ protoplasts/ml in STC.Excess protoplasts were stored in a Cryo 1° C. Freezing Container(Nalgene, Rochester, N.Y., USA) at −80° C.

Approximately 100 μg of transforming plasmid (pSMai214, pDM287, orpEJG107) were digested with Pme I. The digestion reaction was purifiedby 0.8% agarose gel electrophoresis in TAE buffer. A DNA band containingthe expression cassette of pSMai214, pDM287, or pEJG107, and theAspergillus nidulans acetamidase (amdS) gene, was excised from the geland extracted using a NUCLEOSPIN® Extract II Kit according to themanufacturer's suggested protocol.

The resulting purified DNA [1 μg of the 9.9 kb Pme I digested pDM287(tandem transformation) or 1 μg of the 7.6 kb Pme I digested pEJG107plus 1 μg of the 5.4 kb Pme I digested pSMai214 (co-transformation)] wasadded to 100 μl of the protoplast solution and mixed gently. PEG buffer(250 μl) was added, and the reaction was mixed and incubated at 34° C.for 30 minutes. STC (3 ml) was then added, and the reaction was mixedand then spread onto COVE plates for amdS selection. The plates wereincubated at 28° C. for 6-11 days.

Example 5 Evaluation of Trichoderma reesei Transformants ExpressingAspergillus Fumigatus CEL3A Beta-Glucosidase and Aspergillus fumigatusGH 61B Polypeptide

Trichoderma reesei transformants (Example 4) were transferred from COVEtransformation plates to COVE2 plates supplemented with 10 mM uridineusing an inoculation loop and incubated 5-7 days at 28° C. Spores werecollected with an inoculating loop and transferred to 25 ml of CIMmedium in a 125 ml plastic shake flask. The shake flask cultures wereincubated for 5 days at 28° C., 200 rpm. A 1 ml aliquot of each culturewas centrifuged at 13,400×g in a microcentrifuge and culture supernatantwas recovered. Five μl of each culture supernatant were analyzed bySDS-PAGE using a CRITERION® 8-16% Tris-HCl Gel (Bio-Rad Laboratories,Hercules, Calif., USA) according to the manufacturer's instructions. Theresulting gel was stained with BIO-SAFE™ Coomassie (Bio-RadLaboratories, Hercules, Calif., USA). FIGS. 4A-4D show the SDS-PAGEprofiles of the cultures of 45 transformants of pDM287 (tandemconstruct; FIGS. 4A and 4B) and 45 transformants of pEJG107+pSMai214(co-transformation; 4C and 4D). The results demonstrated that thetransformants produced major protein bands of approximately 130 kDacorresponding to the A. fumigatus CEL3A beta-glucosidase andapproximately 24 kDa corresponding to the A. fumigatus GH61Bpolypeptide. A negative control sample, consisting of untransformed T.reesei strain 981-O-8 (D4) culture supernatant, showed no prominentbands at approximately 130 kDa and approximately 24 kDa.

The results in FIGS. 4A-4D and summarized below demonstrated thattransformation with the tandem construct pDM287 yielded more positivetransformants for A. fumigatus beta-glucosidase and A. fumigatus GH61Bpolypeptide production than co-transformation with pEJG107 and pSMai214.

Number of transformants positive for A. fumigatus beta-glucosidaseTransforming and A. fumigatus GH61B polypeptide DNA production bySDS-PAGE pDM287 (tandem construct) 33 of 45 (73%) pEJG107 + pSMai214 13of 45 (29%) (co-transformation)

Example 6 Beta-Glucosidase Assay of Trichoderma reesei TransformantsExpressing Aspergillus Fumigatus CEL3A Beta-Glucosidase and Aspergillusfumigatus GH61B Polypeptide

The culture supernatants of Example 5 were assayed for beta-glucosidaseactivity using a BIOMEK® 3000, a BIOMEK® NX, and an ORCA® robotic arm(Beckman Coulter, Inc, Fullerton, Calif., USA). Culture supernatantswere diluted appropriately in 0.1 M succinate, 0.01% TRITON® X-100(4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) buffer pH 5.0(sample buffer) followed by a series of dilutions from O-fold to ⅓-foldto 1/9-fold of the diluted sample. A total of 20 μl of each dilution wastransferred to a 96-well flat bottom plate. Two hundred microliters of ap-nitrophenyl-beta-D-glucopyranoside substrate solution (1 mg ofp-nitrophenyl-beta-D-glucopyranoside per ml of 0.1 M succinate pH 5.0)were added to each well and then incubated at ambient temperature for 45minutes. Upon completion of the incubation period 50 μl of quenchingbuffer (1 M Tris buffer pH 9) were added to each well. An endpoint wasmeasured at an optical density of 405 nm for the 96-well plate.

The results shown in FIG. 5 confirmed the SDS-PAGE results of Example 5that transformation with the tandem construct pDM287 yielded morepositive transformants for A. fumigatus beta-glucosidase and A.fumigatus GH61B polypeptide production than co-transformation withpEJG107 and pSMai214.

The present invention is further described by the following numberedparagraphs:

[1] A method for obtaining positive transformants of a filamentousfungal host cell, comprising: (a) transforming into a population ofcells of the filamentous fungal host a tandem construct comprising (i)one or more selectable markers, (ii) a first polynucleotide encoding afirst polypeptide having biological activity operably linked to a firstpromoter and a first terminator, and (iii) a second polynucleotideencoding a second polypeptide having biological activity operably linkedto a second promoter and a second terminator; (b) selectingtransformants based on the one or more selectable markers, wherein thenumber of positive transformants for the first and second polypeptideshaving biological activity obtained by transformation of the tandemconstruct is higher compared to the number of positive transformantsobtained by co-transformation of separate constructs for each of thefirst and second polynucleotides; and (c) isolating a transformant ofthe filamentous fungal host cell comprising the tandem constructexpressing the first and second polypeptides having biological activity.

[2] The method of paragraph 1, wherein the number of positivetransformants for the first and second polypeptides having biologicalactivity obtained by transformation of the tandem construct is increasedat least 1.1-fold, e.g., at least 1.25-fold, at least 1.5-fold, at least2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least5-fold, or at least 10-fold, compared to the number of positivetransformants obtained by co-transformation of separate constructs foreach of the first and second polynucleotides.

[3] The method of paragraph 1 or 2, wherein the tandem constructintegrates by ectopic integration into the chromosome of the filamentousfungal host cell.

[4] The method of any of paragraphs 1-3, wherein the tandem construct iscontained in an expression vector.

[5] The method of any of paragraphs 1-4, wherein the tandem constructfurther comprises a first homologous repeat flanking 5′ of the one ormore selectable markers and a second homologous repeat flanking 3′ ofthe one or more selectable markers, wherein the first homologous repeatand the second homologous repeat undergo homologous recombination toexcise the one or more selectable markers.

[6] The method of paragraph 5, wherein the first and second homologousrepeats are identical or have a sequence identity of at least 70%, e.g.,at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 97%, atleast 98%, or at least 99% to each other.

[7] The method of paragraph 5 or 6, wherein the first and secondhomologous repeats are each at least 50 bp, e.g., at least 100 bp, atleast 200 bp, at least 400 bp, at least 800 bp, at least 1000 bp, atleast 1500 bp, or at least 2000 bp.

[8] The method of any of paragraphs 5-7, wherein upon the excision ofthe one or more selectable markers, the one or more selectable markerscan be reused for introducing another tandem construct into thefilamentous fungal host cell.

[9] The method of any of paragraphs 1-8, wherein the polypeptides havingbiological activity are different polypeptides.

[10] The method of any of paragraphs 1-8, wherein the polypeptideshaving biological activity are the same polypeptide.

[11] The method of any of paragraphs 1-10, wherein the promoters aredifferent promoters.

[12] The method of any of paragraphs 1-10, wherein the promoters are thesame promoter.

[13] The method of any of paragraphs 1-12, wherein the terminators aredifferent terminators.

[14] The method of any of paragraphs 1-12, wherein the terminators arethe same terminator.

[15] The method of any of paragraphs 1-14, wherein the filamentousfungal cell is an Acremonium, Aspergillus, Aureobasidium, Bjerkandera,Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus,Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora,Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete,Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, □Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

[16] The method of paragraph 15, wherein the Trichoderma strain isselected from the group consisting of Trichoderma harzianum, Trichodermakoningii, Trichoderma longibrachiatum, Trichoderma reesei, andTrichoderma viride.

[17] The method of paragraph 15, wherein the Trichoderma strain isTrichoderma reesei.

[18] A filamentous fungal host cell, comprising: a tandem constructcomprising (i) one or more selectable markers, (ii) a firstpolynucleotide encoding a first polypeptide having biological activityoperably linked to a first promoter and a first terminator, and (iii) asecond polynucleotide encoding a second polypeptide having biologicalactivity operably linked to a second promoter and a second terminator.

[19] The filamentous fungal host cell of paragraph 18, wherein thetandem construct integrated by ectopic integration into the chromosomeof the filamentous fungal host cell.

[20] The filamentous fungal host cell of paragraph 18 or 19, wherein thetandem construct is contained in an expression vector.

[21] The filamentous fungal host cell of any of paragraphs 18-20,wherein the tandem construct further comprises a first homologous repeatflanking 5′ of the one or more selectable markers and a secondhomologous repeat flanking 3′ of the one or more selectable markers,wherein the first homologous repeat and the second homologous repeatundergo homologous recombination to excise the one or more selectablemarkers.

[22] The filamentous fungal host cell of paragraph 21, wherein the firstand second homologous repeats are identical or have a sequence identityof at least 70%, e.g., at least 75%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 97%, at least 98%, or at least 99% to each other.

[23] The filamentous fungal host cell of paragraph 21 or 22, wherein thefirst and second homologous repeats are each at least 50 bp, e.g., atleast 100 bp, at least 200 bp, at least 400 bp, at least 800 bp, atleast 1000 bp, at least 1500 bp, or at least 2000 bp.

[24] The filamentous fungal host cell of any of paragraphs 21-23,wherein upon the excision of the one or more selectable markers, the oneor more selectable markers can be reused for introducing another tandemconstruct into the filamentous fungal host cell.

[25] The filamentous fungal host cell of any of paragraphs 18-24,wherein the polypeptides having biological activity are differentpolypeptides.

[26] The filamentous fungal host cell of any of paragraphs 18-24,wherein the polypeptides having biological activity are the samepolypeptide.

[27] The filamentous fungal host cell of any of paragraphs 18-26,wherein the promoters are different promoters.

[28] The filamentous fungal host cell of any of paragraphs 18-26,wherein the promoters are the same promoter.

[29] The filamentous fungal host cell of any of paragraphs 18-28,wherein the terminators are different terminators.

[30] The filamentous fungal host cell of any of paragraphs 18-28,wherein the terminators are the same terminator.

[31] The filamentous fungal host cell of any of paragraphs 18-30,wherein the filamentous fungal cell is an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, □ Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

[32] The filamentous fungal host cell of paragraph 31, wherein theTrichoderma strain is selected from the group consisting of Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, and Trichoderma viride.

[33] The filamentous fungal host cell of paragraph 31, wherein theTrichoderma strain is Trichoderma reesei.

[34] A method of producing multiple recombinant polypeptides havingbiological activity, comprising: cultivating a filamentous fungal hostcell transformed with a tandem construct comprising (i) one or moreselectable markers, (ii) a first polynucleotide encoding a firstpolypeptide having biological activity operably linked to a firstpromoter and a first terminator, and (iii) a second polynucleotideencoding a second polypeptide having biological activity operably linkedto a second promoter and a second terminator, under conditions conducivefor production of the polypeptides.

[35] The method of paragraph 34, further comprising recovering the firstand second polypeptides having biological activity.

[36] The method of paragraph 34 or 35, wherein the tandem constructintegrated by ectopic integration into the chromosome of the filamentousfungal host cell.

[37] The method of any of paragraphs 34-36, wherein the tandem constructis contained in an expression vector.

[38] The method of any of paragraphs 34-37, wherein the tandem constructfurther comprises a first homologous repeat flanking 5′ of the one ormore selectable markers and a second homologous repeat flanking 3′ ofthe one or more selectable markers, wherein the first homologous repeatand the second homologous repeat undergo homologous recombination toexcise the one or more selectable markers.

[39] The method of paragraph 38, wherein the first and second homologousrepeats are identical or have a sequence identity of at least 70%, e.g.,at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 97%, atleast 98%, or at least 99% to each other.

[40] The method of paragraph 38 or 39, wherein the first and secondhomologous repeats are each at least 50 bp, e.g., at least 100 bp, atleast 200 bp, at least 400 bp, at least 800 bp, at least 1000 bp, atleast 1500 bp, or at least 2000 bp.

[41] The method of any of paragraphs 37-40, wherein upon the excision ofthe one or more selectable markers, the one or more selectable markerscan be reused for introducing another tandem construct into thefilamentous fungal host cell.

[42] The method of any of paragraphs 34-41, wherein the polypeptideshaving biological activity are different polypeptides.

[43] The method of any of paragraphs 34-41, wherein the polypeptideshaving biological activity are the same polypeptide.

[44] The method of any of paragraphs 34-43, wherein the promoters aredifferent promoters.

[45] The method of any of paragraphs 34-43, wherein the promoters arethe same promoter.

[46] The method of any of paragraphs 34-45, wherein the terminators aredifferent terminators.

[47] The method of any of paragraphs 34-45, wherein the terminators arethe same terminator.

[48] The method of any of paragraphs 34-47, wherein the filamentousfungal cell is an Acremonium, Aspergillus, Aureobasidium, Bjerkandera,Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus,Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora,Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete,Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, □Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

[49] The method of paragraph 48, wherein the Trichoderma strain isselected from the group consisting of Trichoderma harzianum, Trichodermakoningii, Trichoderma longibrachiatum, Trichoderma reesei, andTrichoderma viride.

[50] The method of paragraph 48, wherein the Trichoderma strain isTrichoderma reesei.

[51] A tandem construct comprising (i) one or more selectable markers,(ii) a first polynucleotide encoding a first polypeptide havingbiological activity operably linked to a first promoter and a firstterminator, and (iii) a second polynucleotide encoding a secondpolypeptide having biological activity operably linked to a secondpromoter and a second terminator.

[52] The tandem construct of paragraph 51, wherein the tandem constructfurther comprises a first homologous repeat flanking 5′ of the one ormore selectable markers and a second homologous repeat flanking 3′ ofthe one or more selectable markers, wherein the first homologous repeatand the second homologous repeat undergo homologous recombination toexcise the one or more selectable markers.

[53] The tandem construct of paragraph 52, wherein the first and secondhomologous repeats are identical or have a sequence identity of at least70%, e.g., at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 97%, at least 98%, or at least 99% to each other.

[54] The tandem construct of paragraph 52 or 53, wherein the first andsecond homologous repeats are each at least 50 bp, e.g., at least 100bp, at least 200 bp, at least 400 bp, at least 800 bp, at least 1000 bp,at least 1500 bp, or at least 2000 bp.

[55] The tandem construct of any of paragraphs 52-54, wherein upon theexcision of the one or more selectable markers, the one or moreselectable markers can be reused for introducing another tandemconstruct into the filamentous fungal host cell.

[56] The tandem construct of any of paragraphs 51-55, wherein thepolypeptides having biological activity are different polypeptides.

[57] The tandem construct of any of paragraphs 51-55, wherein thepolypeptides having biological activity are the same polypeptide.

[58] The tandem construct of any of paragraphs 51-57, wherein thepromoters are different promoters.

[59] The tandem construct of any of paragraphs 51-57, wherein thepromoters are the same promoter.

[60] The method of any of paragraphs 51-59, wherein the terminators aredifferent terminators.

[61] The method of any of paragraphs 51-59, wherein the terminators arethe same terminator.

[62] An expression vector comprising the tandem construct of any ofparagraph 51-61.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

What is claimed is:
 1. A method for obtaining positive transformants ofa filamentous fungal host cell, comprising: (a) transforming into apopulation of cells of the filamentous fungal host a tandem constructcomprising (i) one or more selectable markers, (ii) a firstpolynucleotide encoding a first polypeptide having biological activityoperably linked to a first promoter and a first terminator, and (iii) asecond polynucleotide encoding a second polypeptide having biologicalactivity operably linked to a second promoter and a second terminator,wherein the tandem construct integrates by ectopic integration into thechromosome of the filamentous fungal host cell; (b) selectingtransformants based on the one or more selectable markers, wherein thenumber of positive transformants for the first and second polypeptideshaving biological activity obtained by transformation of the tandemconstruct is higher compared to the number of positive transformantsobtained by co-transformation of separate constructs for each of thefirst and second polynucleotides; and (c) isolating a transformant ofthe filamentous fungal host cell comprising the tandem constructexpressing the first and second polypeptides having biological activity.2. The method of claim 1, wherein the number of positive transformantsfor the first and second polypeptides having biological activityobtained by transformation of the tandem construct is increased at least1.1-fold compared to the number of positive transformants obtained byco-transformation of separate constructs for each of the first andsecond polynucleotides.
 3. The method of claim 1, wherein the tandemconstruct further comprises a first homologous repeat flanking 5′ of theone or more selectable markers and a second homologous repeat flanking3′ of the one or more selectable markers, wherein the first homologousrepeat and the second homologous repeat undergo homologous recombinationto excise the one or more selectable markers.
 4. The method of claim 3,wherein the first and second homologous repeats are identical or have asequence identity of at least 70% to each other.
 5. The method of claim3, wherein upon the excision of the one or more selectable markers, theone or more selectable markers can be reused for introducing anothertandem construct into the filamentous fungal host cell.
 6. The method ofclaim 1, wherein the number of positive transformants for the first andsecond polypeptides having biological activity obtained bytransformation of the tandem construct is increased at least 2-foldcompared to the number of positive transformants obtained byco-transformation of separate constructs for each of the first andsecond polynucleotides.
 7. The method of claim 1, wherein the number ofpositive transformants for the first and second polypeptides havingbiological activity obtained by transformation of the tandem constructis increased at least 5-fold compared to the number of positivetransformants obtained by co-transformation of separate constructs foreach of the first and second polynucleotides.
 8. The method of claim 1,wherein the number of positive transformants for the first and secondpolypeptides having biological activity obtained by transformation ofthe tandem construct is increased at least 10-fold compared to thenumber of positive transformants obtained by co-transformation ofseparate constructs for each of the first and second polynucleotides. 9.The method of claim 3, wherein the first and second homologous repeatshave a sequence identity of at least 80% to each other.
 10. The methodof claim 3, wherein the first and second homologous repeats have asequence identity of at least 90% to each other.
 11. The method of claim3, wherein the first and second homologous repeats are each at least 50bp.
 12. The method of claim 1, wherein the polypeptides havingbiological activity are different polypeptides.
 13. The method of claim1, wherein the polypeptides having biological activity are the samepolypeptide.
 14. The method of claim 1, wherein the promoters aredifferent promoters.
 15. The method of claim 1, wherein the promotersare the same promoter.
 16. The method of claim 1, wherein theterminators are different terminators.
 17. The method of claim 1,wherein the terminators are the same terminator.
 18. The method of claim1, wherein the filamentous fungal cell is an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phiebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.
 19. The method of claim 18, wherein theTrichoderma strain is selected from the group consisting of Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, and Trichoderma viride.
 20. The method of claim 18,wherein the Trichoderma strain is Trichoderma reesei.